Protocol 45710: Probe Assay - Best3<em1(IMPC)J>
Version 1.0

Notes

Taqman qPCR protocols are run on a real time PCR instrument. Use an appropriate instrument specific Fluorophore/Quencher combination.

 

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

>chr10:117002332+117002427 96bp ACGTGTGCACCTTGTATGTG GGTAGGGGAGGGTGCTAAAG

Mutant=  96 bp

Wild Type = 96 bp

Sequence

Wt Sequence: acgtgtgcaccttgtatgtgcacgtgcacacacatacActtgggtgcataacacccggaggcaagaatctttacttctttagcaccctcccctacc

Mut Sequence: acgtgtgcaccttgtatgtgcacgtgcacacacatacACttggaagtatttgctgtctgcacagcacgctcttccagaggctgatgggaaaagtgt

 

A 469 bp deletion beginning at Chromosome 10 position 117,002,370 bp and ending after 117,002,838 bp (GRCm38/mm10).

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
66270 ACG TGT GCA CCT TGT ATG TG Common A
66271 GGT AGG GGA GGG TGC TAA AG Wild type Reverse A
66272 ACA CTT TTC CCA TCA GCC TCT Mutant Reverse A
66273 Fluorophore-1 TGC ACA CAC ATA CAC TTG GAA GTA T Quencher-1 MUT Probe
66274 Fluorophore-2 CAT AAC ACC CGG AGG CAA Quencher-2 WT Probe

Reaction A

Component Final Concentration
Kapa Probe Fast QPCR 1.00 X
ddH2O
66270 0.40 uM
66271 0.40 uM
66272 0.40 uM
Wt Probe 0.15 uM
Mutant Probe 0.15 uM
DNA

Cycling

Step Temp °C Time Note
1 95.0 --
2 95.0 --
3 60.0 --
4 -- repeat steps 2-3 for 40 cycles
5 40.0 -- Forever
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.

Strains Using This Protocol

This is the only strain that uses this protocol.